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  • Author: H. KANAGAWA x
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Anatomical and histological examinations on three intersex pigs revealed that two of these carried bilateral ovotestes, well-developed uterine horns and female-like external genitalia, while the third had rudimentary male accessory sex glands and unequal uterine horns with an ovary attached to the right horn and a testis to the left. Chromosome analyses on blood and testicular tissue of the female-like intersexes showed exclusively XX cells whereas the intersex with male accessory sex glands exhibited a small proportion of XY cells in cultures of blood, testis and kidney, suggesting that the latter may be a whole body chimera. In contrast to these cattle freemartins, intersex pigs, including the two with ovotestes, carried apparently functional ovaries. The escape from virilization of the female gonads, despite their proximity to testes in intersex pigs, may be attributable to the biological nature of the testicular secretion which, in boars, is rich in sulphated oestrogens.

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Different types of secretory cells were observed in the cervical mucosa of the uteri of macaques and rabbits. The percentage of ciliated cells in the macaque (10%) and rabbit (62%) varies in different regions of the cervix. Kinocilia, observed in vitro, beat towards the vagina. The beating of cervical cilia does not seem to be of importance for the transport of spermatozoa to the uterine cavity. Scanning electron microscope studies indicate that kinocilia may be of functional significance for the release into the cervical lumen of material from the surface of adjacent secretory cells in the cervical crypts.

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A. A. Rayos, Y. Takahashi, M. Hishinuma and H. Kanagawa

Unfertilized mouse oocytes were frozen by directly plunging them into liquid nitrogen vapour after equilibration in a freezing medium containing 3 mol ethylene glycol l−1 with 0.25 mol sucrose or trehalose l−1 for 5–40 min. After thawing and dilution of the cryoprotectant, oocytes of normal morphology were inseminated in vitro and the effect of equilibration period on the rates of fertilization and development in vitro was examined. Regardless of the equilibration in the freezing medium, no significant difference was observed on the fertilization rate of frozen–thawed oocytes. However, higher fertilization and higher normal fertilization rates were obtained with equilibration in 3 mol ethylene glycol l−1 with either 0.25 mol sucrose l−1 or trehalose for 20 and 40 min than with 5 and 10 min equilibration. Development rates to two-cell embryos and expanded blastocysts of in vitro fertilized frozen–thawed oocytes that were equilibrated in the freezing medium for 20 and 40 min were significantly higher (P < 0.05 or P < 0.01) than with 5 min equilibration. Development in vivo was assessed by transferring blastocysts derived from unfertilized oocytes frozen by the optimum treatment (20 min equilibration in the freezing medium before freezing) into the uterine horns of day 3 pseudopregnant female recipients. The development rate of frozen–thawed oocytes to the blastocyst stage after insemination in vitro was significantly lower than that of the non-frozen control (P < 0.001). However, transfer of the blastocysts derived from frozen-thawed oocytes to the uterine horns of the recipients resulted in fetal development and implantation rates similar to those of the control. The overall development rates to fetuses of blastocysts derived from in vitro fertilization of mouse oocytes frozen after 20 min equilibration in 3 mol ethylene glycol l−1 with 0.25 mol sucrose l−1 or trehalose were 20.3 and 22.5%, respectively.

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C. A. Valdez, O. Abas Mazni, Y. Takahashi, S. Fujikawa and H. Kanagawa

Summary. Mouse blastocysts were exposed to solutions containing four concentrations (10, 20, 30 and 40% v/v) of six permeating cryoprotectants (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide, 1,3-butanediol and 2,3-butanediol) in phosphate-buffered saline (PBS) with calf serum (CS) at room temperature (20–22°C). Blastocysts were exposed to these solutions for various periods, diluted into PBS plus CS with or without 1 mol trehalose l−1 solution and their subsequent survival in vitro was examined. Two-way anova showed a significant interaction (P < 0·01) between cryoprotectant type, concentration of cryoprotectant and method of dilution. However, no significant interaction was observed between cryoprotectant type and duration of exposure. Results suggest that cryoprotectant-induced injury to nonfrozen blastocysts is variable and depends on the cryoprotectant used. On the basis of toxicity assays, ethylene glycol was the least harmful and was combined with dimethyl sulfoxide and 1,3-butanediol to produce a new vitrification solution. Mouse blastocysts were successfully cryopreserved using a vitrification solution (designated as VSv) consisting of 20% ethylene glycol, 20% dimethyl sulfoxide and 10% 1,3-butanediol (v/v). Embryos were equilibrated in two steps, first in an equilibration solution (designated as ESv: 10% ethylene glycol, 10% dimethyl sulfoxide and 5% 1,3-butanediol; v/v) and then to VSv or one-step in VSv at different exposure times at room temperature, and then vitrified by direct plunging into liquid nitrogen. High developmental rates were obtained in vitro when the embryos were exposed to ESv and VSv for 3 and 0.5 min, respectively (96·2%) or exposed to VSv for 0·5 min (95·4%). Prolonged exposure time proved detrimental to subsequent embryo development in vitro. When vitrified warmed embryos were transferred immediately to pseudopregnant recipients, the rate of development to normal fetuses did not significantly differ from that of the nonvitrified control (two-step, 54·2 and one-step, 45·0 versus 60·0%, P > 0·05).

These results suggest that the simple vitrification solution described in this study is effective for the cryopreservation of mouse blastocysts.

Keywords: mouse; blastocyst; vitrification; cryoprotectant; equilibration